Optical cell and methods of manufacturing an optical cell

ABSTRACT

An optical cell for performing light spectroscopy (including absorbance, fluorescence and scattering measurements) on a liquid sample in microfluidic devices is disclosed. The optical cell comprises an inlaid sheet having an opaque material inlaid in a clear material, and a sensing channel that crosses the clear material and the opaque material provides a fluidic path for the liquid sample and an optical path for probe light. Integral optical windows crossing a clear-opaque material interface permit light coupling into and out of the sensing channel, and thus light transmission through the sensing channel is almost entirely isolated from background light interference. A microfluidic chip comprising one or more optical cells is also disclosed. The optical cells may have different lengths of sensing channels, and may be optically and fluidly coupled. A method of manufacturing an optical cell in a microfluidic chip is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/854,602, filed on May 30, 2019, the entire contentsof which is incorporated herein by reference for all purposes.

TECHNICAL FIELD

The present disclosure relates to light spectroscopy for analyzing aliquid sample, and in particular to an optical cell for performing lightspectroscopy.

BACKGROUND

Microfluidic technologies manipulate small amounts of fluid withinchannels for resource-efficient processes. Lab-on-a-chip (LOC) systemsuse microfluidic platforms to miniaturize standard analysis techniques,integrating one or several laboratory functions onto one device. Thesedevices combine as many bench top fluid analysis methods as possible toa single, small-scale, low-power chip. Reduced sample sizes enables morecost-, energy-, and reagent-efficient analyses that produce less waste.

Furthermore, these devices may be automated to reduce—or eveneliminate—the needed level of human involvement in environmentalsampling and analysis. With in situ analysis capabilities, conventionalrisks of sample contamination during transport from field to lab areremoved and collected data are more reliable. Possibilities of humanerror are reduced, and hazardous environments or chemicals may behandled with minimal risk to human life. The generally small form factorof microfluidic systems allows them to be less environmentally invasivethan other methods, and their smaller sample sizes mean that they arealso less destructive. Furthermore, they have the potential to domeasurements at high pressures, which has been useful for prolongedsensor deployment in ocean environments.

A microfluidic absorbance cell may utilize screwed-in or epoxy-fixedfibers to couple light to/from a flow cell for sample inspection.However, fiber-based designs tend to be less transferrable from the labto the field, where slight mechanical shocks/vibrations can result inreduced optical coupling and sensor measurement error. Alternatively,low-cost robust optical absorbance measurements may be realized by usingthe chip material itself. A completely integrated absorbance cell inchips fabricated from carbon-doped (tinted) semi-transparent PMMA hasbeen developed, however it is based on a challenging alignment andfabrication process and requires either UV-curable or time settingtwo-part epoxies to completely immerse the LED and photodiode to holdthem in perfect alignment to the microchannel. This requires a skilledand tedious workflow during the manufacturing process of these devices,where measurements must be taken to ensure optical alignment.

Accordingly, an optical cell and methods of manufacturing thereof remainhighly desirable.

SUMMARY

In accordance with one aspect of the present disclosure, an optical cellfor performing light spectroscopy on a liquid sample is disclosed,comprising: an inlaid sheet comprising an opaque material inlaid withina clear material; a sensing channel having first and second ends andproviding a fluidic path for the liquid sample and an optical path forprobe light between the first and second ends, wherein the sensingchannel crosses the clear material and the opaque material; an opticalinput opening coupled with the first end of the sensing channel fordelivering the probe light to the sensing channel; and at least oneoptical detection opening coupled with the sensing channel for receivingthe probe light having interacted with the liquid sample.

In some aspects, the at least one optical detection opening comprises afirst optical detection opening coupled to the sensing channel proximatethe first end.

In some aspects, the sensing channel has a well structure with increasedfluid volume where the first optical detection opening is coupled to thesensing channel.

In some aspects, the at least one optical detection opening comprises asecond optical detection opening coupled with the second end of thesensing channel.

In some aspects, the optical cell further comprises fluid inlet andoutlet ports respectively coupled with the first and second ends of thesensing channel.

In some aspects, the optical cell further comprises fluid inlet andoutlet channels arranged in the clear material and respectively coupledwith the fluid inlet and outlet ports.

In some aspects, the fluid inlet and outlet channels are coupled to thefluid inlet and outlet ports at at least one angle of 135 degrees orless relative to the sensing channel.

In some aspects, the fluid inlet and outlet channels are coupled to thefluid inlet and outlet ports at opposing sides of the sensing channel.

In some aspects, the optical input opening and the at least one opticaldetection opening extend from a surface of the layer of clear materialto a depth of the sensing channel.

In some aspects, the optical input opening comprises an input prismdisposed in the optical input opening at the depth of the sensingchannel for directing the probe light into the sensing channel.

In some aspects, an optical detection opening of the at least oneoptical detection opening comprises an output prism disposed in theoptical detection opening at the depth of the sensing channel fordirecting the probe light out of the optical cell.

In some aspects, the optical input opening and the at least one opticaldetection opening are surrounded by the opaque material.

In some aspects, the optical cell further comprises a light guidechannel extending within the inlaid sheet and coupling the optical inputopening and the first end of the sensing channel.

In some aspects, the opaque material has approximately 0% lighttransmittance.

In some aspects, the clear material has greater than approximately 90%light transmittance.

In some aspects, one or both of the opaque material and the clearmaterial are configured to filter selected wavelengths.

In some aspects, the sensing channel has a length between 0.1 mm and 100mm.

In some aspects, the inlaid sheet comprises first and second inlaidsheets bonded together, the first inlaid sheet comprising a first opaquematerial inlaid in a recess of a first layer of clear material, and thesecond inlaid sheet comprising a second opaque material inlaid in arecess of a second layer of clear material; and wherein the sensingchannel is provided at an interface of the first and second inlaidsheets.

In accordance with another aspect of the present disclosure, amicrofluidic cell is disclosed, comprising the optical cell of any ofthe above aspects.

In accordance with another aspect of the present disclosure, amicrofluidic cell is disclosed, comprising a plurality of optical cellsof any of the above aspects.

In some aspects, the plurality of optical cells are fluidly coupled toeach other such that the liquid sample flows through the plurality ofoptical cells in series.

In some aspects, the plurality of optical cells are optically coupled toeach other via optical reflectors.

In some aspects, a total optical path length of the plurality of opticalcells is up to 10 m.

In some aspects, a length of the sensing channel of at least two opticalcells is different, the length of each of the sensing channels beingbetween 0.1 mm and 100 mm.

In accordance with another aspect of the present disclosure, a methodfor manufacturing an optical cell in a microfluidic chip is disclosed,comprising: cutting first and second inserts of an opaque material;cutting recesses in first and second sheets of clear material forrespectively receiving the first and second inserts of the opaquematerial; inserting the first and second inserts of opaque material intothe recesses of the first and second sheets of clear material to formfirst and second inlaid sheets; cutting a sensing channel along abonding surface of at least one of the first and second inlaid sheets,the sensing channel crossing the clear material and the opaque material;and bonding the first and second inlaid sheets together.

In some aspects, the method further comprises defining an optical inputopening and at least one optical detection opening in the first layer ofclear material, wherein the optical input opening is coupled with afirst end of the sensing channel, and wherein the at least one opticaldetection opening is coupled with the sensing channel.

In some aspects, the method further comprises cutting a prism in theoptical input opening at a depth of the sensing channel.

In some aspects, an optical detection opening of the at least oneoptical detection opening is coupled with a second end of the sensingchannel, and the method further comprises cutting a prism in the opticaldetection opening at a depth of the sensing channel.

In some aspects, the first insert of the opaque material surrounds theoptical input opening and the at least one optical output opening.

In some aspects, the method further comprising cutting a light guidechannel extending along the bonding surface of at least one of the firstand second inlaid sheets, wherein the light guide channel couples theoptical input opening and the sensing channel.

In some aspects, the method further comprises cutting fluid inlet andoutlet ports respectively coupled with first and second ends of thesensing channel.

In some aspects, the method further comprises cutting fluid inlet andoutlet channels coupled to the fluid inlet and outlet ports.

In some aspects, the opaque material has approximately 0% lighttransmittance.

In some aspects, the clear material has greater than approximately 90%light transmittance.

In some aspects, one or both of the opaque material and the clearmaterial are configured to filter selected wavelengths.

In some aspects, the sensing channel has a length between 0.1 mm and 100mm.

In some aspects, a cross-section of the sensing channel is between 0.01mm and 1 mm.

In some aspects, the first and second sheets of clear material are asingle sheet of clear material, and wherein the first and second inlaidsheets are cut from the single sheet.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1A shows an exploded cross-sectional view of a microfluidic chipcomprising an inlaid optical cell;

FIG. 1B shows a perspective view of a microfluidic chip comprising anoptical cell;

FIGS. 2A and 2B show a representation of light passing through anoptical cell;

FIG. 3 shows a configuration of a plurality of optical cells havingdifferent path lengths on a chip;

FIG. 4 shows a method of manufacturing an optical cell;

FIG. 5 shows a fabrication process of manufacturing a microfluidic chipcomprising the optical cell;

FIG. 6 shows raw photodiode voltage vs. time of typical (a) yellow fooddye, (b) nitrite, and (c) phosphate experiments through the inlaidoptical cell;

FIG. 7 shows absorbance versus concentration for (a) red food dye andfor (b) reacted nitrite samples;

FIG. 8 shows absorbance versus concentration for (a) yellow food dye andfor (b) reacted phosphate samples; and

FIGS. 9 and 10 show fluorescence test data conducted with the inlaidoptical cell.

It will be noted that throughout the appended drawings, like featuresare identified by like reference numerals.

DETAILED DESCRIPTION

The present disclosure provides a new technique for implementing lightspectroscopy in microfluidic devices in which optically-isolated cellsare created within clear materials. This technique creates what isreferred to herein as an inlaid optical cell, combining clear and opaquematerials into one fluidly-sealed device. In accordance with the presentdisclosure, an optical cell is disclosed that combines clear and opaque(˜0% transmission) to completely attenuate background light withoutdiminishing source intensity. This technique also introduces thepossibility of directing light in/out of the chip using integratedv-groove prisms (which is difficult to achieve using chips made of oneuniform tinted material, for example).

An optical cell for performing light spectroscopy (including absorbance,fluorescence and scattering measurements) on a liquid sample inmicrofluidic devices is disclosed. The optical cell comprises an inlaidsheet having an opaque material inlaid in a clear material, and asensing channel that crosses the clear material and the opaque materialprovides a fluidic path for the liquid sample and an optical path forprobe light. Integral optical windows crossing a clear-opaque materialinterface permit light coupling into and out of the sensing channel, andthus light transmission through the sensing channel is almost entirelyisolated from background light interference. The design of the opticalcell allows for manipulating small (micro-litre to femto-litre scale)amounts of fluid within channels of dimensions on the micro-meter scale.The inlaid optical cell does not rely on epoxies to align or hold thesource and detector, and advantageously allows for changing the lightsource and detector combination for different chemistries on the samechip design without detaching optics/epoxy fixatives that would riskdamaging the microfluidic chip. This design is highly manufacturable, asrepeatable and robust optical alignment can be achieved through standardmanufacturing practices of component placement on printed circuit boards(PCBs).

A microfluidic chip comprising one or more optical cells is alsodisclosed. The optical cells may have different lengths of sensingchannels, and may be optically and fluidly coupled. A method ofmanufacturing an optical cell in a microfluidic chip is also disclosed.

Light-absorbance measurements are enabled by transmitting light from anLED through various fluids contained in a sensing channel to alight-to-voltage converter (photodiode) for detection. To couple lightinto and out of the channel, the optical cell as disclosed hereinemploys prisms, as further described with reference to FIG. 1. Accordingto Snell's law, light of wavelength λ, will undergo total internalreflection (TIR) at the cell-air prism interface, so long as the angleof incidence θ_(i) exceeds the critical angle θ_(c):

$\begin{matrix}{\theta_{c} = {\sin^{- 1}\left( \frac{n_{2}(\lambda)}{n_{1}(\lambda)} \right)}} & (1)\end{matrix}$

where n₁(λ) and n₂(λ), the refractive indices of the cell material andair, are functions of the light wavelength. Where poly(methylmethacrylate) (PMMA) is used as the cell material, the angle ofinterface may for example be 45° to the incident beam on both prisms.

Absorbance spectroscopy is a common and well-established technique forbiological and chemical analysis, either in continuous or stopped-flowconfigurations. It is a simple, quick, and robust method of detectingthe presence of a species within a fluid sample provided the analyteconcentration is relatively low and that it has an accessible/uniqueabsorbance spectrum. Incident light passing through a sample willattenuate depending on the molecular absorbance spectrum of the sample,the scattering from particles in the fluid, and the spectrum of incidentlight. For molecular absorbance, the molecules within the analyte maybecome excited and thus absorb energy from the incident waves aselectrons are promoted to higher energy bands. The relative drop inlight energy—or equivalently its intensity—can be used to calculate theabsorbance of the sample using the Beer-Lambert law,

$\begin{matrix}{{A = {{- {\log_{10}\left( \frac{I}{I_{0}} \right)}} = {\in {lc}}}},} & (2)\end{matrix}$

where absorbance A depends on the ratio of incident to transmitted lightintensities, I₀ and I, respectively. The intensities are used to findthe concentration c of the absorbing species as a function of theoptical path length l and the attenuation coefficient of the analyte ϵ.Here, the effects of scattering attenuation can be ignored as the fluidsamples are sufficiently filtered. Significantly, the detectionsensitivity has a linear dependence on the length of the opticalchannel: a challenge for microfluidic devices that aim to minimizephysical size.

Interference from ambient background light—light which reaches thedetector without passing through the sample—must be quantified orotherwise minimized. Equation (2) may be modified to eliminate theseexternal influences and is expressed in the following equivalent forms:

$\begin{matrix}{{A_{corr} = {{- {\log_{10}\left( \frac{I - I_{bg}}{I_{0} - I_{bg}} \right)}} = {{A + {\log_{10}\left( \frac{1 - {I_{bg}/I_{0}}}{1 - {I_{bg}/I}} \right)}} = {A + {\delta\mspace{14mu}\left( {\delta \geq 0} \right)}}}}},} & (3)\end{matrix}$

where I_(bg) is the intensity of background light measured by thedetector when the light source is turned off. This background intensityis subtracted off each sample and blank measurement to calculate thecorrected absorbance of the sample A_(corr). The corrected absorbance isalways greater or equal to the uncorrected absorbance A since ∀I_(bg)≤0,δ≥0. This follows directly from the fact that I₀≥I since light intensityis at its maximum when passing through a colorless blank. In practice,however, Schlieren mixing effects and refractive index mismatches thatare thermal/composition based can lead to this being not true.

Furthermore, it can be proved that higher-concentrated samplesexperience more severe corrections than lower-concentrated samples.Sample concentration and the detected intensity of light that has passedthrough them, I, are negatively or inversely related. Evaluating thechange in δ with respect to I while holding all other variablesconstant, we see:

$\begin{matrix}{\frac{\partial\delta}{\partial I} = {{\frac{1}{\ln\; 10} \times \frac{I_{bg}}{I\left( {I_{bg} - I} \right)}} < 0.}} & (4)\end{matrix}$

Equation (4) is always negative since 0≤I_(b)<I, indicating a negativerelationship between I and δ. As stated above, sample concentration andI are also negatively related, and so it follows that sampleconcentration and δ are positively related and higher-concentratedsamples experience larger corrections. In other words, the amount oflight detected is less for highly concentrated samples, and therefore,any background light detected has a greater influence on the absorbance.Detection of background light must therefore be quantified for everysample, particularly when highly concentrated samples are beingmeasured.

UV/vis spectroscopy is well suited for microfluidic platforms, andbiosensors, as an example, may use UV/vis OAS to determineconcentrations of nutrients or other biological entities within samplesor in situ within sections of an environment. Microfluidic absorptionspectroscopy may be used for analyzing biofilms, studying singlemolecules in enzymatic reactions, detecting the presence of specificbacteria species, cellular phenotyping, detecting changes in cellmorphology and many other applications. Beyond biosensing, microfluidicsensors have potential for use in the nuclear industry for activelydetermining actinide concentrations produced during the PUREX process,pharmaceutical development, and even detecting pollutants produced bydental rinses.

The present disclosure introduces a new inlaid approach of an opticalcell for performing light spectroscopy, and is capable of performing notonly absorption measurements but also fluorescence and scattering. Theoptical cell may be particularly useful in marine environments. Theworld's oceans have thus far been grossly under-sampled. Traditionalapproaches to sensing in these environments are labour-, time-, andresource-intensive, depending on manually-collected bottle samples fromthe ocean for analysis far-away in a lab. Consequently, much about theworld's oceans is unknown. With a goal of developing cheap,easy-to-produce, nutrient sensors that can be mass-deployed in theoceans, the performance of the inlaid optical cells disclosed herein isdemonstrated. Low-power, low-contact microfluidic sensors have thepotential to remedy these issues and provide much needed data about thestate of the oceans. A person skilled in the art will also appreciatethat the optical cell as disclosed herein is not limited to suchapplications, and that the optical cell may be used in other fieldswithout departing from the scope of this disclosure.

Embodiments are described below, by way of example only, with referenceto FIGS. 1-10.

Reference is made concurrently to FIGS. 1A and 1B and FIGS. 2A and 2B.FIG. 1A shows an exploded cross-sectional view of a microfluidic chip100 comprising an optical cell. FIG. 1B shows a perspective view of amicrofluidic chip comprising an optical cell. FIGS. 2A and 2B show arepresentation of light passing through an optical cell.

The optical cell comprises a first inlaid sheet 102 and a second inlaidsheet 104 that are bonded together and which define a sensing channel106 there-between. The sensing channel 106 has first and second ends andprovides a fluidic path for a liquid sample and an optical path forprobe light between the first and second ends. The sensing channel 106may have various lengths, but is generally between 0.1 mm and 100 mmdepending on the application. A characteristic cross-section of thesensing channel 106 may be between 10 micrometers and 1 mm.

Each of the first and second inlaid sheets 102, 104, comprise a clearmaterial 102 a, 104 a, and an opaque material 102 b, 104 b inlaid in theclear material 102 a, 104 a. Specifically, the clear material 102 a, 104a comprises recesses (see, e.g., recess 108 in the bottom clear material104 a) that the opaque material 104 b is inserted into. The opaquematerial 102 b, 104 b has approximately 0% light transmittance such thatonly light that travels within the sensing channel persists, while theclear material 102 a, 104 a has greater than approximately 90% lighttransmittance. Moreover, one or both of the opaque material and theclear material may be configured to filter selected wavelengths, whichenables integral optical filtering within the microfluidic device. Atleast one of the first and second inlaid sheets 102, 104 (in FIG. 1A,the first inlaid sheet 102) comprises a cut-out in a bonding surfacethereof that defines the sensing channel 106 when bonded with the otherinlaid sheet (in FIG. 1A, when bonded with the second inlaid sheet 104).

Note that while FIG. 1A shows the optical cell comprising first andsecond inlaid sheets 102, 104 bonded together, the optical cell couldinstead be formed from a single inlaid sheet comprising an opaquematerial inlaid within a clear material. The single inlaid sheet may forexample be made from a 3D printer that prints hybrid materials. In thiscase, the sensing channel may be provided within the inlaid sheet,instead of enclosed by two inlaid sheets bonded together.

The inlaid sheets 102 and 104 may for example be made from a plasticsuch as PMMA. PMMA sheets are typically manufactured in one of two ways,each with different bonding behavior. The first, cast PMMA, is producedby casting liquid methyl methacrylate monomer into a mould made of glassfor polymerization. The second, extruded PMMA, is produced in sheets ofmore uniform thicknesses by shaping melted acrylic monomers with a die.Extruded PMMA is the type of acrylic that is exemplary used in certainembodiments herein, though inlaid optical channels may be created usingboth types. Other common materials used in microfluidic devicefabrication could also be used, including but not limited to:poly-carbonate (PC), poly-propylene (PP), poly-sulfone (PS, PSU, PSUL),Polyetherimide (PEI, Ultem), SU-8 photocurable epoxy, Norland opticalepoxies, various laminates, fluorinated ethylene propylene (FEP), cyclicolefin copolymer (COC), and glasses such as borosilicate, sodalime, etc.

The optical cell further comprises an optical input opening 110 coupledwith a first end of the sensing channel 106 for delivering the probelight to the sensing channel, and one or more optical detection openings(in FIGS. 1 and 2, optical detection openings 112 a-b) that are coupledwith the sensing channel 106 and which receive probe light havinginteracted with the liquid in the sensing channel 106. A light guidechannel 116 may extend within the inlaid sheet and couple the opticalinput opening 110 and the first end of the sensing channel 106. In use,a light source such as an LED may be placed over the optical inputopening 110. The light guide channel 116 may help to isolate a lightdetector from the light source by adding distance between the two. Theoptical detection openings may be provided for obtaining different typesof measurements on the liquid sample. For example, the first opticaldetection opening 112 a, which is coupled to the sensing channel 106proximate a first end of the sensing channel where the probe light isincident to the channel, may be used for performing fluorescencemeasurements and/or scattering measurements. For performing fluorescencemeasurements, a filter may be provided to filter certain wavelengths.The second optical detection opening 112 b is coupled to the second endof the sensing channel 106 where probe light having interacted with theliquid sample exits the sensing channel 106. The second opticaldetection opening 112 b may be used for performing absorptionmeasurements, for example, by placing a photodiode or spectrometer abovethe second optical detection opening 112 b. A benefit of this opticalcell is the ability to use light sources and detectors external to thechip. By coupling both to a printed circuit board (PCB) located directlyabove the cell, near-instant signal processing may occur with minimalwiring.

Note that while first and second optical detection openings 112 a-b areshown, the optical cell may only comprise optical detection opening 112a or optical detection opening 112 b. If there is no optical detectionopening 112 b coupled to the second end of the sensing channel 106, anoptical reflector such as a mirror may for example be placed at thesecond end of the sensing channel 106 to reflect the probe light backthrough the sensing channel 106 and out through the optical inletopening 110.

As described in more detail herein, the optical input opening 110 andthe optical detection openings 112 a-b may be formed by cutting into theclear material (such as clear material 102 a) from a surface thereof toa depth of the sensing channel 106. The recesses of the clear material102 a, and the shape of the opaque material 102 b inserted into theclear material 102 a, is configured such that the openings aresurrounded circumferentially by the opaque material 102 b (see, e.g.,FIG. 1A). The sensing channel 106, cut into the inlaid sheet 102, thuscrosses between the clear material 102 a at the openings and the opaquematerial 102 b elsewhere, and is thus primarily enclosed by the opaquematerial 102 b and 104 b. This helps to ensure that light is transmittedfrom one side of the sensing channel 106 to the other almost exclusivelythrough the sensing channel.

While the figures depict the optical input opening 110 and the opticaldetection openings 112 a-b as being vertical, it is noted that theopenings may also be cut into the material at an angle (such as +/−45degrees to vertical). The optical input opening 110 and the opticaldetection opening 112 b may further comprise v-shaped prisms 114 cutinto the clear material for directing the probe light into the sensingchannel 106 and out of the optical cell. As previously described, theprisms 114 direct the light using TI R, and may be set at an angledependent upon the cell material and the wavelength of incident light.This approach allows for freedom of source/detector location.

The optical cell may further comprise fluid inlet and outlet portsrespectively coupled with the first and second ends of the sensingchannel 106 (see, e.g., fluid port 118 cut in the opaque material 102 bin FIG. 1A coupled with the second end of the sensing channel 106). Acorresponding port may be cut into the clear material in the opticaldetection opening 112 b for coupling to the sensing channel 106. Theoptical cell may further comprise fluid inlet and outlet channels 120arranged in the clear material (for example, clear material 102 a) andrespectively coupled with the fluid inlet and outlet ports. The fluidthus crosses between the clear material 102 a and the opaque material102 b. The fluid inlet and outlet channels 120 may receive fluid fromvias 122, for example. Continuous flow systems can also be deployed,injecting small volumes into a continuous sample stream and loweringvolume per sample measurement to picoliter or femtoliter levels

The fluid inlet and outlet channels 120 may be coupled to the fluidinlet and outlet ports 118 at an angle of 135 degrees or less relativeto the sensing channel 106. The fluid inlet and outlet channels 120 mayalso be coupled to the fluid inlet and outlet ports 118 at opposingsides of the sensing channel 106. For example, as shown in FIG. 1B, thefluid inlet and outlet channels 120 and the sensing channel 106 may forma z-shape pattern. The location where the fluid inlet and outletchannels 120 and the sensing channel 106 meet may thus form an elbowjoint. The fluid inlet and outlet channels 120 may also be coupled tothe fluid inlet and outlet ports at an angle relative to horizontal.

Furthermore, the sensing channel 106 may have a well structure 122 alonga length thereof with increased fluid volume where the first opticaldetection opening 112 a is coupled to the sensing channel 106. The wellstructure 122 is perhaps best seen in FIG. 1B, and may for example beconfigured as a section of sensing channel 106 with a largercircumference or perimeter relative to the remainder of the sensingchannel 106. As described in more detail with reference to FIG. 9,providing the well structure 122 may help to improve detection of afluorescence signal.

Various modifications may also be made to the design of the optical cellwithout departing from the scope of this disclosure. As one example,side-scatter and back-scatter optical channels can also be integratedinto the microfluidic device based on this inlaying technique. A fluidwith particles (cells, suspended matter, etc.) flowing through theoptical channel could be illuminated from the absorbance LED and opticalchannels at various pre-defined angles and would enable particle-sizingand sample characterization.

FIG. 3 shows a configuration of a plurality optical cells havingdifferent path lengths on a chip. As further described with reference toFIGS. 4 and 5, the optical cell 100 may be readily integrated into amicrofluidic chip for performing fluid analysis. Moreover, a pluralityof optical cells, including optical cells of different path lengths, maybe integrated onto the same chip.

FIG. 3 shows a first optical cell 300 a having a 50 mm sensing channeland a second optical cell 300 b having a 25 mm sensing channel disposedon the same microfluidic chip. An LED 302 is shown as being positionedover the opening at one end of the optical cell 300 a, and a transmittedbeam is shown as being emitted from the other end of the optical cell300 a.

As evident from FIG. 3, a microfluidic cell may comprise a plurality ofoptical cells, which may for example have sensing channels of differentlengths. The plurality of optical cells may be fluidly coupled to eachother such that the liquid sample flows through the plurality of opticalcells in series. Further, the plurality of optical cells may beoptically coupled to each other via optical reflectors. A total fluidpath length and optical path length may thus be greatly increasedrelative to a length of a single sensing channel, and may for examplereach lengths of up to 10 m by way of serially coupling many opticalcells. Furthermore, two optical cells of different path lengths may beconfigured in an “L-shape” or “180-degree shape” sharing an illuminationprism (i.e. the light from a single LED reflects down both cells) withseparate detectors prisms.

Reference is made concurrently to FIG. 4 and FIG. 5 for manufacturingthe optical cell as part of a microfluidic chip. FIG. 4 shows a methodof manufacturing an optical cell. FIG. 5 shows a fabrication process ofmanufacturing a microfluidic chip comprising the optical cell.

Two inserts are cut from opaque material to be inserted into recesses ofclear material (402). Corresponding recesses are cut in clear sheets ofmaterial for respectively receiving the inserts made of opaque material(404). The recesses in the clear material are of matching dimensions totheir corresponding insert except with an added tolerance such as 25 μmon all sides, which helps to compensate for microscopic deformities andimprecise milling so that the inserts fit tightly within the recesses.As previously described, the materials used may be PMMA or othersmentioned above. The opaque material may have approximately 0% lighttransmittance, and the clear material may have greater thanapproximately 90% light transmittance. Moreover, one or both of theopaque material and the clear material may be configured to filterselected wavelengths.

As seen in FIG. 5, a sheet of clear material 502 comprises recesses 504a and 504 b, for respectively receiving inserts of opaque material 506 aand 506 b. Two separate sheets of clear material could instead be used.The length of the inserts are dependent in part upon the length of thesensing channel, which may for example range from between 0.1 mm and 100mm. As also seen in FIG. 5, cutting the recesses in the clear materialmay further comprise defining ridges for openings to be cut extendingpartially through the material in what will become a first layer of theclear material in the assembled optical cell, and the correspondingopaque insert 506 a may be shaped with holes to completely surround theopenings. There may be an optical input opening and at least one opticaldetection opening cut into the clear material. The second opaque insert506 b has the same shape as the first insert 506 a, but does notcomprise the holes extending there-through so as to block incident lightnot directed into the channel as described with reference to FIG. 1.

The opaque inserts 506 a-b are pressed into the recesses 504 a-b of theclear material and bonded to form first and second inlaid sheets (406).In FIG. 5, first and second inlaid sheets 508 a-b may be cut from thesheet of clear material after insertion of the opaque inserts 506 a-b.The inserts may be bonded with the recesses of the clear sheets with asolvent, thermal, and pressure process. To achieve a fluid-tight sealbetween the two plastics that ensures uncompromised fluid handling,solvent depolymerization then repolymerization may be performed.

Prior to bonding, a pre-treatment may be performed for each surface.Pre-treatment included sanding rough edges with a fine-grit paper andlight scrubbing such as with a scotch pad sponge with water anddetergent. A brush can be used to scrub inside the cavities. A rinse,such as with Milli-Q may be conducted, and surfaces may be dried withblasts of compressed air or lab-grade nitrogen gas and IPA. Afterpre-treatment, substrates were ready for bonding. Chloroform may bepreheated to 30° C. in a sealed container or petri dish.Chloroform-vapor exposure is performed by suspending each substrate 2 mmabove the chloroform liquid line, face down for 45 seconds. Afterchloroform exposure, the inserts were quickly slotted into the cavitiesand pressed for approximately half a minute. Next, the clear sheet withthe inlay ensemble was pressed for 2.5 hours in an LPKF Multipress IIset to a pressure of 625 N/cm² and a temperature of 115° C. to approachthe glass transition temperature of PMMA. After pressing, minorprotrusion of the opaque inserts may be observed from the sheet. Thearea that would encompass the entire chip design may be milled down by asmall amount, approximately 0.3 mm, to restore uniformity and preventdelamination when bonding the top and bottom layers together laterduring the fabrication.

A sensing channel is created along a bonding surface of at least one ofthe first/second inlaid sheets (408). In addition, other components suchas a light guide channel, inlet/outlet fluidic channels, alignmentholes, vias, syringe ports, and prisms, may be created (410). These canbe milled into the appropriate spots within the inlaid sheet to realizethe microfluidic design. FIG. 5 shows sensing channel 510 cut into thebonding surface (i.e. a bottom surface of the top layer) of the inlaidsheet 508 a, which is cut between two openings in the clear material andextends through the opaque insert. A light guide channel may also be cutthat extends along the bonding surface of the first/second inlaid sheetthat couples the optical input opening and the sensing channel. Thefirst insert of the opaque material and openings in the clear materialmay also be cut to define fluid inlet and outlet ports respectivelycoupled with first and second ends of the sensing channel. Fluid inletand outlet channels may be cut in the first layer of the clear materialthat are coupled to the fluid inlet and outlet ports of the first insertof the opaque material. Prisms may be cut in the optical input openingand/or the optical detection opening at a depth of the sensing channel.

The fluidic channels may be milled depending on application parametersbut may for example be approximately 400 μm wide and 600 μm deep and bemilled into the top layer using a corresponding mill bit. The prisms maybe created by cutting into the top layer using a 45-degree end-mill to aspecified depth. FIG. 5 shows an example of first and second inlaidsheets 508 a-b with various features created therein.

The first and second inlaid sheets are bonded (412). Prior to bonding,the first and second inlaid sheets may be cut to a desired size, andleft for approximately 12 hours heated to a temperature of approximately85° C. to release residual stress and/or solvent vapor trapped withinthe inlay. The first and second inlaid sheets may be bonded togetherusing a 45 seconds chloroform-vapor exposure time and similar bondingand pressing parameters as above but with a slightly lower bondingtemperature such as 85° C. FIG. 5 shows a microfluidic chip 512 formedfrom the first and second inlaid sheets 508 a-b.

As previously described, a microfluidic chip may comprise a plurality ofoptical cells formed therein. As would be evident to a person skilled inthe art, in the method for manufacturing described above the first andsecond inlaid sheets may be prepared for forming multiple optical cellsby cutting a plurality of opaque inserts and corresponding recesses inthe clear material.

To validate the optical cell, two nutrients fundamental to aquaticecosystems were measured: nitrite and phosphate, over three differentlengths of sensing channels: 10, 25, and 50 mm. Light-absorbancemeasurements of nitrite samples reacted with the “Griess” reagent isconsidered a gold-standard approach. Under acidic conditions, a purpleazo dye with peak absorbance near 540 nm will form in the presence ofnitrite. Phosphate detection is enabled using an acidic ammoniummolybdate/metavanadate based reagent, hereby referred to as phosphatereagent, which forms a yellow complex proportional to phosphateconcentration with maximum absorbance in the UV region at 375 nm. Withboth chemistries, the inlaid microfluidic absorbance cell as disclosedherein produced results in agreement with literature and are thusacceptable to be used for automated in situ sensors. Other phosphatechemistries (EPA 365.2) that absorb in the 880 nm range are alsopossible with an infrared LED. Similarly, nitrate measurement can beperformed using established Vanadium-reduction that yield nitrite to bereacted with Griess reagent, thereby enabling nitrate measurement withthe same optical system. Nitrite on its own can also be measured usingthe Griess reagent without vanadium reduction

Performance of the absorbance cell was first characterized with twodifferent colors of food dye that had peak absorbances near those ofreacted nitrite and phosphate. Red food dye standards were prepared bytwo-fold serial dilutions of a 0.1% stock, made from dilution of 0.1 mLred food dye (commercial food coloring, Club House Canada) to 100.0 mLwith Milli-Q water. Yellow food dye standards were similarly made fromdiluting a 0.1% stock, made in the same way (commercial food coloring,Club House Canada). Nitrite standards were prepared via stepwisedilution of a 1000 11M stock, mixed from 0.069 g of sodium nitrite(NaNO2, CAS-No: 7632-00-0, EMD Millipore, Germany) and Milli-Q to atotal volume of 1 L. Phosphate standards were prepared similarly from a1000 11M stock, produced by diluting 0.1361 g of potassium phosphatemonobasic (KH2PO4, BP362-500 LOT 184646, Fisher Scientific) to 1 L withMilli-Q. Standards were stored in darkness near room temperature betweenuse.

The reagents were prepared so that reagent molecules were in excess ofexpected nitrite/phosphate ions to ensure color-development isproportional to concentration. The Griess reagent was prepared bycombining 0.5 g of sulfanilamide, 5 mL of concentrated HCl, and 0.05 gof NEDD (N-(1-Naphthyl)ethylenediamine dihydrochloride), mixed withMilli-Q to a final volume of 500 mL. Finally, phosphate reagent wasprepared by mixing 0.3601 g of ammonium metavanadate and 7.2 g ofammonium molybdate with 95 mL of concentrated HCl, and brought to afinal volume of 1 L with Milli-Q. Constant stirring ensured propermixing and dissociation of any precipitates formed, and the reagent wasallowed to cool to room temperature after the exothermic reaction uponaddition of HCl. Chemistry components were of analytical grade, andcompleted reagents were stored near 4° C. in a dark environment betweenuse.

Two LEDs centered at Δ₁=527 nm (Cree C503B-GAN-CB0F0791-ND, FWHM=15 nm)and Δ₂=380 nm (Superbrightleds RL5-UV0315-380, FWHM=12 nm) were used toperform absorbance spectroscopy on reacted nitrite and phosphatesamples—and their corresponding food dye samples—respectively withdetection by a Digikey TSL257 High-Sensitivity Light-to-VoltageConverter (photodiode). A custom-built LED driver allowed adjustment ofLED intensity while maintaining constant current. The voltage output ofthe photodiode was connected to a B&K Precision 5491B bench multimeterwith 10⁻⁷ V precision. The sampling rate was set to its maximum, andeach measurement along with their date and time was recorded on apersonal laptop connected to the multimeter via USB.

LED intensity was adjusted with Milli-Q in the channel to maximize lightdetection without saturating the photodiode. Since sources and detectorswere held externally to the chip, a single set of optical components(LED and photodiode) were used for all three path lengths and the entiretesting process. To inspect a sample using one of the three opticalcells, the relevant LED was held in place directly above a prism using ametal clamp and a photodiode was held face-down above the prism on theother end. Swapping between each of the three optical cells or changingLEDs between test series was simple due to the decoupled components: auseful advantage of this optical system. A custom opaque enclosure wasplaced over the entire testing apparatus to minimize background lightreception.

Fluid injection for the red food dye and nitrite tests was performedmanually with syringes. Yellow food dye and phosphate tests werecompletely automated using an off-the-self Cavro XC syringe pump (PN20740556-C, Tecan Systems, San Jose, Calif.) and a Vici Cheminert C65Z10-port selector valve (Model No. C65-37101A, Valco Instruments Co.Inc., Houston Tex.). Each of the four inspected species were analyzedusing all three optical channels a total of three times each. At thestart, end, and between each sample, Milli-Q was pumped through thechannel to flush the system and reduce sample-sample crosstalk. Thisalso served as a blank measurement for all but the phosphate testswherein a 1:1 volumetric mix of Milli-Q and reagent was used instead toserve as the blank since the inherent color of the reagent itself was alight-yellow. All samples and blanks were analyzed for a period of fiveminutes; the average voltage over the final minute was used to determineabsorbance. Nitrite samples were analyzed immediately following reactionwith the Griess reagent whereas phosphate samples were premixed severalhours before analysis permitting complete reaction. Finally, at thebeginning and end of every test, a measurement of the background lightwas taken over a minute interval. These values were usually very smallrelative to the blank reading and were averaged together for use withequation (3) to account for background light. Other mixing ratios couldbe used, depending on the chemistry utilized.

FIG. 6 shows raw photodiode voltage vs. time of typical (a) yellow fooddye, (b) nitrite, and (c) phosphate experiments through a 10.4 mm inlaidoptical cell. The dye plot in (a) depicts an entire triplicate withlabels normalized to the highest concentration sample: 0.1%. The nitriteand phosphate plots depict a single trial within a triplicate, andlabels indicate the concentration of the associated standard beforemixing with reagent. Blanks are analyzed between successive samples.

The performance of the inlaid optical cell design was first evaluatedwith stable food dyes before analyzing nutrient samples. Food dyecalibration tests are a standard benchmark towards proving robust,accurate, and reliable light-absorbance measurements of an optical cell.There are several external factors that may influence thelight-absorbance of a reacted nutrient sample with reagent. Theseinclude sample degradation, reagent degradation, incorrect sample orreagent make-up, and the kinetics of the reaction between nutrient andreagent. In the case where samples do not produce the expectedresults—i.e. conforming to the Beer-Lambert law given by equation (2)—itis difficult to determine if the design of the optical cell is at faultor the analyzed samples themselves. Red dye was chosen to mimic reactednitrite due to its absorbance spectrum with a strong absorbance peak inthe same 540 nm region. Using the 527 nm LED, light-absorbancemeasurements were taken through red dye samples ranging from 0.0016 to0.05% (v/v). Similarly, yellow food dye was chosen due to mimic reactedphosphate where the peak absorbance spectrum was 375 nm and inspectedusing the 380 nm LED A range of yellow dye was tested between0.0063%-0.1% (v/v). After successful food dye experiments, nitritesamples were reacted with the Griess reagent and analyzed. Nitritestandards ranged in concentration from 0.1 μM to 100 μM and weremeasured using the 527 nm LED, after mixing in a 1:1 volumetric ratiowith reagent. Therefore, the final concentration of each analyzed samplewas half that of the standard. Finally, phosphate standards between 0.1μM-50 μM were reacted with phosphate reagent and were analyzed using the380 nm LED.

FIG. 6(a) shows five concentrations of yellow food dye analyzed a totalof three times through a 10.4 mm long inlaid optical cell; each seriesproduced consistent results. Milli-Q blanks preceded each sample asdescribed above. Near the 4,000 second mark, a temporary drop in voltagecan be observed: this was likely the result of an air bubble in themeasurement channel during stopped flow. Additional sample was injectedinto the cell to displace the bubble, which restored the expectedvoltage reading as seen before and after this drop. Finally, at the endof the trial, a measurement was taken to quantify the background lightby turning off the LED light source. This is labelled as “Dark Ref.” onFIG. 6(a) and was consistently 7 mV±1 mV throughout experiments in thismanuscript, highlighting the effectiveness of our design in minimizingbackground light contributions.

FIG. 6(b) depicts the results of a nitrite experiment performed using 12standards with Milli-Q blanks between them and with concentrationslabelled on each plateau. Nitrite samples were analyzed immediatelyafter mixing with the Griess reagent. The reaction kinetics can beobserved by the color-development of each reacting sample; i.e. voltageover time. Initially, there was a rapid drop in voltage at the beginningof each reaction followed by a gradual decrease until a plateau wasreached. The lower-concentration nitrite samples, less than 5 μM, appearto have completely reacted almost immediately, attaining 95% of theplateau value within 15 seconds. The higher-concentration nitritesamples, near 50 μM, took longer to react and required 44 seconds toattain 95% of the plateau value.

FIG. 6(c) shows the results of a phosphate experiment performed with 8standards. Here, the blank was a 1:1 volumetric mix of Milli-Q andphosphate reagent, which was injected between standards. The sequence ofinjection was blank, sample and then pure Milli-Q water. The Milli-Qwater flush was used as a precaution to minimize crosstalk betweenstandards. The Milli-Q flush can be observed in FIG. 6(c) as suddenvoltage spikes which saturate the photodiode since the pure Milli-Q iscolorless compared to the blank (LED intensity is set based on blank).For the phosphate experiments, samples were fully reacted prior toinjection into the optical cell. The yellow method has similardevelopment times to the Griess method: 1-5 minutes. The phosphate waspre-reacted to benchmark stable nutrient samples; hence, the relativelyconstant voltage plateaus. In FIG. 6(c), a minor drift of the blankvoltage is observed, decreasing 100 mV over the 6,000 second duration ofthe experiment, which was less than a 2% drop over 1.5 hours. The yellowdye experiment in FIG. 6(a) also drifted down, 70 mV over 6,000 seconds,but this is less visible because of the y-axis scale. The drift wasrepeatedly observed for the phosphate and yellow dye measurements,indicating it is not the phosphate chemistry. The gradual voltagedecrease between blanks is most likely due to UV-fluid interaction orageing of the PMMA plastic through prolonged intense UV-exposure.

FIG. 7 shows absorbance versus concentration for (a) red food dye andfor (b) reacted nitrite samples. The nitrite concentration representsthe final concentration in the flow cell after mixing with Griessreagent. The absorbance of each sample concentration is the average fromthree experiments with vertical error bars representing standarddeviations. Linear fits are shown with R2>0.99, as expected by equation(2).

Light-absorbance of each species was characterized using three inlaidmicrofluidic cells with optical path lengths of 10.4 mm, 25.4 mm, and50.4 mm, labelled as 10 mm, 25 mm, and 50 mm in the legend,respectively. In practice, shorter optical channels are used to detecthighly concentrated samples, but they are less sensitive (smaller slopein Beer-Lambert law). Longer channels are used for detecting smallvariations and low concentrations but are also more susceptible to noisefrom bubbles and particulates. Each calibration curve depicted in FIG. 7represents the average of three independent experiments, except the 10.4mm nitrite series where only two trials were performed. Equation (3) wasused to calculate the absorbance of each sample from their associatedphotodiode readings, referenced to the voltage of the prior blank andthe voltage produced from background light. Linear trendlines withforced-zero intercepts were fit to each series with strong agreement tothe data.

In FIG. 7(a), each optical cell (10, 25 and 50 mm) was evaluated withfour to six different concentrations depending on the path length. The10 mm path length was evaluated with six samples and maintained linearresults consistent with equation (2), even for the most concentratedsamples. The 25 mm path length cell showed a linear relationship for thefirst five samples. The 50 mm path length showed a linear relationshipfor the first four samples. FIG. 7(b) depicts the absorbance of variousconcentrations of reacted nitrite samples. The final concentration ofnitrite after mixing with reagent is used to reflect the trueconcentration in the absorbance cell. All twelve standards were analyzedusing the 10 mm cell; whereas, the least-concentrated ten and ninestandards were analyzed with the 25 mm and 50 mm cells, respectively.For the concentrations tested, it is clear that the inlaid opticalabsorbance cells showed excellent linear relationships.

FIG. 8 shows absorbance versus concentration for (a) yellow food dye andfor (b) reacted phosphate samples. The absorbance of each sampleconcentration is the average from three experiments with vertical errorbars representing standard deviations. Linear fits are shown withR2>0.99, as expected by equation (2).

FIG. 8(a) depicts five yellow food dye samples analyzed with the 10 mmand 25 mm optical path lengths, and four samples analyzed on the 50 mmoptical path length. All three data sets showed excellent linear fitsfor their entire concentration range. Similarly, in FIG. 8(b), theabsorbance for all eight phosphate standards show the expected linearrelationships.

Table 1 shows experimentally determined attenuation coefficients c forboth red and yellow dyes, nitrite, and phosphate for all three opticalpath lengths tested. ϵ is the average with standard deviation. σ_(dye)is volume concentration such that σ_(dye)=v_(dye)/v_(solution). Thesequantities are specific to the inspection light wavelengths, i.e. 527 nmfor red food dye and reacted nitrite, and 380 nm for yellow food dye andreacted phosphate. An average attenuation coefficient of nitrite wasfound to be ϵ(NO₂ ⁻)=0.0269 (μM cm)⁻¹. The value is in agreement withliterature values for ϵ(NO₂ ⁻), which range between 0.014-0.039 (μMcm)⁻¹ for Griess reagent and light centered near 525 nm. Similarly, anaverage attenuation coefficient for phosphate was found to be c (PO₄³⁻)=0.00335 (μM cm)⁻¹. This value is also in agreement with literaturevalues that range between 0.0036-0.00503 (μM cm)⁻¹ for the yellow methodusing light centered near 380 nm. For all three path lengths, theattenuation coefficients determined for nitrite and phosphate usingthese color-developing techniques agree with the literature whichfurther supports the performance of this optical cell.

TABLE 1 Red Food Dye Nitrite Yellow Food Dye Phosphate l ϵ_(RFD) ϵ_(NO)ϵ ϵ (mm) (σ_(dye) cm)⁻¹ ϵ (μM cm)⁻¹ ϵ (σ_(dye) cm)⁻¹ ϵ (μM cm)⁻¹ ϵ 10.41168 1100 0.0273 0.0269 386.1 383 0.00330 0.00335 25.4 1068 ± 0.0264 ±377.9 ± 0.00349 ± 50.4 1081 50 0.027 0.0005 384.24 4 0.00328 0.00012

The limit-of-detection (LOD) can be a useful measure of a sensingapparatus' measuring capabilities and is specific to each speciesmeasured. The resolution of the system can be quantified by measuringthe average noise of n blanks. The LOD for both nitrite and phosphatewas evaluated for each optical path length using the standardtriple-sigma method, which uses three-times the blank baseline noise asa reference. A blank value of 4.80 V—just below photodiodesaturation—was chosen to convert each LOD from voltage to absorbanceunits. The absorbances were then converted to concentrations by dividingby the respective slope for each species. Table 2 shows LODs for nitriteand phosphate for each optical path length; n represents the number ofblanks analyzed, each over five minutes. The slopes depicted in FIG.7(b) and FIG. 8(b) are used to convert absorbance to concentration foreach path length.

TABLE 2 Nitrite Phosphate Avg. Avg. Blank Blank l Noise LOD LOD NoiseLOD LOD (mm) n (mV) (mAU) (nM) n (mV) (mAU) (nM) 10.4 23 3 ± 2 0.9 ± 0.630 ± 20 24 1.3 ± 0.9 0.3 ± 0.3 100 ± 80  25.4 31 4 ± 5 1.0 ± 1.3 14 ± 1920 2 ± 2 0.6 ± 0.6 60 ± 70 50.4 22 3 ± 4 0.7 ± 1.0 6 ± 8 23 2.7 ± 1.80.7 ± 0.5 40 ± 30

These detection limits are consistent with those found in theliterature. A notable trend is that, among each path length, the averagenoise of the nitrite blanks is greater than those of the phosphateblanks: this may be a consequence of the manual sample injection methodused for the nitrite samples. A benefit of automated sample injection ismore consistent injection flow rates between each sample. Although theLODs are 6 nM and 40 nM, the limit-of-quantification (LOQ) would be moreappropriate as a lower sensing bound when these inlaid flow cells areintegrated into marine sensors. Typically, the LOQ is ten times theblank noise, and in this case would be 20 nM and 150 nM for nitrite andphosphate, respectively.

FIGS. 9 and 10 show fluorescence test data conducted with the inlaidoptical cell.

Rhodamine samples ranging in concentration between 1.1 mM-4.7 nM wereprepared from a 0.0104 M Rhodamine B stock solution. Table 3 below showsthe concentration of each sample, numbered in order of preparation. Eachsample was made by pipetting from the stock into 35 mL of Milli-Q waterto achieve the intended concentration.

TABLE 3 Sample Rhodamine Conc. Number (M) 1 1.1E−03 2 2.9E−04 3 2.2E−044 1.5E−04 5 8.9E−05 6 5.9E−05 7 3.0E−05 8 1.5E−05 9 6.0E−06 10 3.0E−0611 1.5E−06 12 6.0E−07 13 3.0E−07 14 1.5E−07 15 7.5E−08 16 3.7E−08 171.9E−08 18 9.3E−09 19 4.7E−09

Rhodamine samples or Milli-Q water blanks were manually injected intothe microfluidic chip via syringe. Samples were excited by directinglight from a 516 nm LED into the fluid channel using prisms. A FlameSpectrometer was used to measure the output spectra to capture anyfluorescence. Milli-Q water was injected through the chip after eachRhodamine sample to flush the system and remove inter-sample crosstalk.Light absorbance measurements are enabled by placing a second detectorover the second prism as described above.

FIG. 9 shows the observed spectra of a 1.5 μM Rhodamine sample asmeasured using two chip designs. Each data set is the average of threemeasurements with error shown in black. Points 904 represent dataobtained using a chip with a rectangular fluid channel (1×1 mmcross-section). Points 902 represent data obtained using the design withan expanded channel comprising the well structure at the measurementwindow as previously described and shown in FIG. 1B. A fluorescencesignal was observed using both designs at ˜577 nm with an improvedsignal observed using the “well” design.

FIG. 10 shows (a) measured spectra of various rhodamine samples ofdiffering concentrations, and (b-c) measured fluorescence plotted vs.rhodamine concentration for each measured sample. Error bars representthe standard deviation of three individual measurements. A log scale isapplied to the x-axis in (c), where more concentrated samples areincluded in the plot to demonstrate quenching effects.

An inlaid optical cell and manufacturing method are thus disclosed inwhich transparent and opaque material are combined to create an isolatedabsorbance cell within a microfluidic chip. Optical components aredecoupled from the chip using integrated v-groove prisms to improvemanufacturability. Light-absorbance measurements were performed usingchannels 400 μm wide and 600 μm deep. Optical path lengths were 10.4 mm,25.4 mm, and 50.4 mm, for total sample volumes ranging between 2.5-12μL. With optimizations, 100-200 μm channels can be achieved and thusnanoliters per measurement can be attained. Samples of varying food dyeconcentration as well as nitrite and phosphate samples were used toverify the measuring capabilities of this novel inlaid approach.Excellent linear relationships were observed between absorbance andconcentration for all tested samples. Further, the capabilities of theoptical cell for performing fluorescence measurements have beendemonstrated.

It would be appreciated by one of ordinary skill in the art that thesystem and components shown in the Figures may include components notshown in the drawings. For simplicity and clarity of the illustration,elements in the figures are not necessarily to scale, are only schematicand are non-limiting of the elements structures. It will be apparent topersons skilled in the art that a number of variations and modificationscan be made without departing from the scope of the invention asdescribed herein.

What is claimed is:
 1. An optical cell for performing light spectroscopyon a liquid sample, comprising: an inlaid sheet comprising an opaquematerial inlaid within a clear material; a sensing channel having firstand second ends and providing a fluidic path for the liquid sample andan optical path for probe light between the first and second ends,wherein the sensing channel crosses the clear material and the opaquematerial; an optical input opening coupled with the first end of thesensing channel for delivering the probe light to the sensing channel;and at least one optical detection opening coupled with the sensingchannel for receiving the probe light having interacted with the liquidsample.
 2. The optical cell of claim 1, wherein the at least one opticaldetection opening comprises a first optical detection opening coupled tothe sensing channel proximate the first end.
 3. The optical cell ofclaim 2, wherein the sensing channel has a well structure with increasedfluid volume where the first optical detection opening is coupled to thesensing channel.
 4. The optical cell of claim 1, wherein the at leastone optical detection opening comprises a second optical detectionopening coupled with the second end of the sensing channel.
 5. Theoptical cell of claim 1, further comprising fluid inlet and outlet portsrespectively coupled with the first and second ends of the sensingchannel.
 6. The optical cell of claim 5, further comprising fluid inletand outlet channels arranged in the clear material and respectivelycoupled with the fluid inlet and outlet ports.
 7. The optical cell ofclaim 6, wherein the fluid inlet and outlet channels are coupled to thefluid inlet and outlet ports at least one angle of 135 degrees or lessrelative to the sensing channel.
 8. The optical cell of claim 6, whereinthe fluid inlet and outlet channels are coupled to the fluid inlet andoutlet ports at opposing sides of the sensing channel.
 9. The opticalcell of claim 1, wherein the optical input opening and the at least oneoptical detection opening extend from a surface of the layer of clearmaterial to a depth of the sensing channel.
 10. The optical cell ofclaim 9, wherein the optical input opening comprises an input prismdisposed in the optical input opening at the depth of the sensingchannel for directing the probe light into the sensing channel.
 11. Theoptical cell of claim 9, wherein an optical detection opening of the atleast one optical detection opening comprises an output prism disposedin the optical detection opening at the depth of the sensing channel fordirecting the probe light out of the optical cell.
 12. The optical cellof claim 1, wherein the optical input opening and the at least oneoptical detection opening are surrounded by the opaque material.
 13. Theoptical cell of claim 1, further comprising a light guide channelextending within the inlaid sheet and coupling the optical input openingand the first end of the sensing channel.
 14. The optical cell of claim1, wherein the opaque material has approximately 0% light transmittance.15. The optical cell of claim 1, wherein the clear material has greaterthan approximately 90% light transmittance.
 16. The optical cell ofclaim 1, wherein one or both of the opaque material and the clearmaterial are configured to filter selected wavelengths.
 17. The opticalcell of claim 1, wherein the sensing channel has a length between 0.1 mmand 100 mm.
 18. The optical cell of claim 1, wherein the inlaid sheetcomprises first and second inlaid sheets bonded together, the firstinlaid sheet comprising a first opaque material inlaid in a recess of afirst layer of clear material, and the second inlaid sheet comprising asecond opaque material inlaid in a recess of a second layer of clearmaterial; and wherein the sensing channel is provided at an interface ofthe first and second inlaid sheets.
 19. A microfluidic cell, comprisingthe optical cell of claim
 1. 20. A microfluidic cell, comprising aplurality of optical cells of claim
 1. 21. The microfluidic cell ofclaim 20, wherein the plurality of optical cells are fluidly coupled toeach other such that the liquid sample flows through the plurality ofoptical cells in series.
 22. The microfluidic cell of claim 20, whereinthe plurality of optical cells are optically coupled to each other viaoptical reflectors.
 23. The microfluidic cell of claim 22, wherein atotal optical path length of the plurality of optical cells is up to 10m.
 24. The microfluidic cell of claim 20, wherein a length of thesensing channel of at least two optical cells is different, the lengthof each of the sensing channels being between 0.1 mm and 100 mm.